This invention relates to a method of determining oxidized .alpha.-1-proteinase inhibitor in serum or plasma. This method is a diagnostic technique useful in studying the development of chronic obstructive lung disease. More specifically this method is useful for patients having a potential for chronic obstructive lung disease rather than using bronchial lavage methods. No other method is known to exist for determining oxidized .alpha.-1-proteinase inhibitor in serum or plasma.
Pulmonary emphysema appears to be a consequence of accelerated lung elastin degradation that results from either an elevated level of elastase activity or a decreased level of elastase inhibitor(s) within the lung, or a combination of both factors. Neutrophil elastase is probably the major elastolytic activity involved in development of emphysema although elastase activity from other sources such as macrophages and monocytes may have a role. The principal inhibitor of neutrophil elastase is the plasma protein .alpha.-1-proteinase inhibitor (formerly referred to as .alpha.-1-antitrypsin). Severe genetic deficiencies of this protein commonly result in emphysema in the affected individuals. .alpha.-1-proteinase prevents the accumulation of leukocytes which contain proteases such as elastase within their cytoplasmic granules.
Most smokers who develop emphysema do so despite normal levels of .alpha.-1-proteinase inhibitor. An explanation for emphysema in smokers with normal .alpha.-1-proteinase inhibitor concentrations has evolved from the observation of the ease of oxidation of the reactive site methionyl residue of this protein leading to a two-thousand fold reduced rate of association of the oxidized inhibitor with human leukocyte elastase. Thus, reduced levels of elastase inhibitory activity can result from partial oxidation of the .alpha.-1-proteinase inhibitor in the lung through inhalation of oxidants present in tobacco smoke, ozone, or industrial gases, as well as from oxidants released from cells in the lung, even if levels of plasma .alpha.-1-proteinase inhibitor are normal or elevated.
Chronic obstructive lung disease has been associated with .alpha.-1-antitrypsin (now referred to as .alpha.-1-proteinase inhibitor) deficiency wherein extraordinarily low levels of elastase inhibition were found in the serum of the survey patients, assaying .alpha.-1-antitrypsin activity by a standard assay known in the art (Turino, G. M., Senior, R. M., Bhagwin, D. C., Keller, S., Levi, M. M., and Mandl, I., Science, 165:709, 1969). Release of proteolytic enzymes in lungs and the onset of emphysema have been linked to cigarette smoking (Hutchinson, D. C. S., Brit. J. Dis. Chest. 67:171, 1973). The relationship of leukocytic proteases to lung tissue degradation and .alpha.-1-antitrypsin inhibitor (now referred to as .alpha.-1-proteinase inhibitor) deficiency has been described which showed a correlation between serum leukocytic elastase inhibitory capacity and serum trypsin inhibitory capacity wherein both trypsin and elastase were inhibited by .alpha.-1-antitrypsin (Lieberman, J., Arch. Environ. Health 27:196, 1973). The proteolytic enzyme elastase has been reported to cause irreversible lung damage after laboratory animals received intratrachial injections of pancreatic porcine elastase (Kaplan, P. D., Kuhn, C., and Pierce, J. A., J. Lab. Clin. Med. 82:349, 1973). The Eriksson (Laurell, C. B. and Eriksson, S., Scand. J. Clin. Lab. Invest. 15:132-134, 1963) method of emphysema diagnosis has been analyzed and an attempt has been made to determine the probability of the occurrence of phenotypes predisposed to emphysema when low .alpha.-1-antitrypsin (now referred to as .alpha.-1-proteinase inhibitor) values were found using gelatinous film or immunochemical methods (Pilacik, B. and Kowalczyk, J., Med. Pr. 30(3):207, 1979). The oxidant effect of cigarette smoking has been reported wherein several mechanisms have been suggested by which tobacco smoke damages the lungs (Kimbel, P. and Kueppers, F., Ann. Intern. Med. 92(4):564, 1980). The concentration of proteases and antiproteases of the lower respiratory tract in normal individuals have been compared with those who smoke or have deficiency of serum .alpha.-1-antitrypsin (now referred to as .alpha.-1-proteinase inhibitor) wherein a mechanism of elastase production within the alveoli of the lung has been suggested (Gadek, J. E., Hunninghake, G. W., Fells, G. A., Zimmerman, R. L., Keogh, B. A., and Crystal, R. G., Bull. europ. Physiopath. resp. 16 (Suppl.):27, 1980). Cigarette smoke has been described as an oxidant of .alpha.-1-proteinase inhibitor wherein as little as three puffs of cigarette smoke caused a significant decrease in the elastase inhibitor capacity per milligram of .alpha.-1-proteinase inhibitor (Janoff, A., Carp, H., and Lee, D. K., Bull. europ. Physiopath. 16 (Suppl.):321, 1980). A cycle has been proposed wherein proteolytic enzymes not only degrade tissue abnormally but also increase the production of oxidants to turn off inhibitor control of proteinases resulting in predicted rapid changes in proteinase levels and proteinase inhibitor levels (Travis, J., Beatty, K., Wong, P. S., and Matheson, N. R., Bull. europ. Physiopath resp. 16 (Suppl.):341, 1980). A gel plate assay for the detection of elastase activity and for the measurement of serum elastase inhibitory capacity has been reported which provides an indirect means for estimating .alpha.-1-antitrypsin (now referred to as .alpha.-1-proteinase inhibitor) function (Billingsly, G. D. and Cox, D. W., Am. Rev. resp. Disease. 121 (1):161-164, 1980).
The present invention describes a convenient method for determining the percent of normal versus oxidized .alpha.-1-proteinase inhibitor in human serum or plasma based on a measurable difference between the inhibitory activities of normal and oxidized .alpha.-1-proteinase inhibitor against a trypsin like enzyme and elastase. It is an object of the present invention to provide a method of determining oxidized .alpha.-1-proteinase inhibitor in serum or plasma.
It is an object of the present invention to provide a method for determining the quantity of normal versus oxidized .alpha.-1-proteinase inhibitor against a trypsin like enzyme and elastase.
It is an object of the present invention to provide a useful diagnostic technique in studying the development of chronic obstructive lung disease.
It is a further object to provide a useful diagnostic technique in studying patients having a potential for chronic obstructive lung disease rather than using bronchial lavage methods.
These and other objects, aspects, and advantages of this invention will become apparent from a consideration of the accompanying specification and claims.